Erythropoietin in thyroid cancer.

Erythropoietin (Epo) and the epo-receptor (EpoR) have been implicated in tumor growth, invasion and metastasis. We previously demonstrated Epo and EpoR expression in a small group of archived papillary thyroid cancers (PTC), but were unable to examine functional integrity using formalin-fixed tissues. In the present study, we examined the in vitro expression, induction and function of Epo and EpoR in papillary (NPA), follicular (WRO) and anaplastic (ARO-81) thyroid cancer cells. We found that all three cell lines expressed Epo and EpoR mRNA and that the hypoxia-mimetic cobalt induced Epo expression in all cell lines. None of the growth factors we examined (thyrotropin, vascular endothelial growth factor, IGF-I, or human Epo) altered Epo or EpoR gene expression. Importantly, however, administration of Epo to NPA but not WRO cells resulted in significant alterations in the expression of several mitogenic genes including cyclooxygenase-2 (COX-2), beta-casein (CSN2), wild type p53-induced gene-1 (WIG1) and cathepsin D (CTSD). Epo treated ARO-81 cells only had an increase in CSN2 expression. We conclude that Epo and EpoR are expressed by thyroid cancers and that stimulation of the Epo/EpoR signal pathway results in changes that could impact on the clinical behavior of thyroid cancers.

Nitrotyrosine, inducible nitric oxide synthase (iNOS), and endothelial nitric oxide synthase (eNOS) are increased in thyroid tumors from children and adolescents.

Nitric oxide (NO) is a reactive cell signal that controls vascular tone and is generated by inducible (iNOS), endothelial (eNOS) and neuronal (nNOS) NO synthase (NOS). We hypothesized that NO could be important for growth of thyroid tumors and tested this hypothesis, by staining 41 papillary thyroid carcinoma (PTC), 9 follicular thyroid carcinoma (FTC), and 15 benign thyroid lesions for iNOS, eNOS and nitrotyrosine (N-TYR). Staining intensity was determined by 2 blinded, independent examiners, and quantified from grade 1 (absent) to grade 4 (intense). Average N-TYR staining of benign adenomas (2.5+/-0.42, p=0.009), PTC (3.10+/-0.12, p=0.001), FTC (2.44+/-0.30, p=0.001), and autoimmune lesions (3.25+/-0.48, p=0.019) were greater than that of multinodular goiter (1.0 for all 3) and surrounding normal thyroid (1.1+/-0.1). Average iNOS staining of benign adenomas (2.6+/-0.37), PTC (2.7+/-0.16), FTC (2.4+/-0.26) and autoimmune lesions (3.5+/-0.29) were all greater than that of surrounding normal thyroid (1.1+/-0.1, p<0.008), but there were too few multinodular goiters to achieve a significant difference (no.=2, 3.0+/-1.0). Average eNOS staining of benign adenomas (2.9+/-0.40), multinodular goiters (3.5+/-0.5), PTC (3.24+/-0.18), FTC (3.5+/-0.50), and autoimmune lesions (2.8+/-0.6) were also greater than that of surrounding normal thyroid (mean=1.4+/-0.2, p<0.001). N-TYR staining correlated with that of vascular endothelial growth factor (VEGF, r=0.36, p=0.007) and the number of lymphocytes/high power field (r=0.39, p=0.004). Recurrent disease developed only from carcinoma with moderate-intense N-TYR staining, but there were too few recurrent tumors to achieve statistical significance (p=0.08). We conclude that NO is produced by benign adenomas, PTC and FTC suggesting that NO could be important in vascularization of thyroid tumors and autoimmune thyroid diseases.

Matrix metalloproteinase (MMP) expression by differentiated thyroid carcinoma of children and adolescents.

The factor(s) that control metastasis of thyroid carcinoma are unknown, but the matrix metalloproteinases (MMPs) are excellent candidates. MMP-1, membrane-type-1 MMP (MT1-MMP), and tissue inhibitor of MMP-1 (TIMP-1) have all been implicated, but the site of production and importance are disputed. In vitro, normal thyroid cells secrete TIMP-1, while thyroid cancer cells secrete TIMP-1 and MMP-1. However, previous pathological studies identified MMP-1 and TIMP-1 only in the stroma surrounding thyroid carcinoma. These data suggest that thyroid carcinoma or tumor-associated inflammatory cells might secrete a factor(s) which stimulates MMP-1 or TIMP-1 expression by surrounding tissues. We hypothesized that MMP-1, MT1-MMP, and TIMP-1 would be directly expressed by thyroid carcinoma and might promote invasion or metastasis. We used immunohistochemistry to determine the expression of MMP-1, MT1-MMP, and TIMP-1 in 32 papillary thyroid carcinoma (PTC), 10 follicular thyroid carcinoma (FTC) and 13 benign thyroid lesions from children and adolescents. The intensity of staining was graded from absent (grade 0) to intense (grade 3). Average MMP-1 expression (mean relative intensity units+/-SE) was significantly greater among PTC (1.97+/-0.15; p=0.004) and FTC (2.2+/-0.25; p=0.006) compared to benign lesions (1.30+/-0.15); but there was no relationship between MMP-1 expression and invasion, metastasis, or recurrence. Expression of MT1-MMP and TIMP-1 was similar for benign and malignant lesions; but recurrent PTC expressed lower levels of TIMP-1 when compared to non-recurrent PTC (p=0.049). Only the expression of TIMP-1 correlated with the presence of tumor-associated lymphocytes (r=0.35, p=0.032). We conclude that MMP-1, MT1-MMP and TIMP-1 are all expressed by thyroid carcinoma and could be important in promoting recurrence.

Thyroid carcinomas that express telomerase follow a more aggressive clinical course in children and adolescents.

With each cell division, DNA is lost from the telomeres, limiting the number of divisions, and leading to senescence. Malignant tumors maintain immortality by expressing a specific DNA repair enzyme, telomerase, that replaces this DNA. We hypothesized that tumors which express telomerase would have the highest recurrence risk and we tested this by determining telomerase expression in 27 papillary thyroid carcinomas (PTC), 5 follicular thyroid carcinomas (FTC) and 13 benign thyroid lesions from children and adolescents. Patients were 6-21 yr of age (mean+/-SE=16.6+/-4.1 yr) and followed from 0-14.1 yr (mean+/-SE=4.71+/-3.5 yr). Original tumors were sectioned, and immunostained for telomerase. Telomerase-specific staining was determined by two independent, blind examiners and graded from absent (Grade 0) to intense (Grade 3). Telomerase was detected in a similar majority of benign (11/13, 85%) and malignant tumors (24/32, 75%). However, the intensity of telomerase expression was greater among FTC (mean+/-SE=2.4+/-0.5 relative intensity) followed by PTC (mean+/-SE=1.9+/-1.0 relative intensity) and benign tumors (mean+/-SE=1.8+/-1.0 relative intensity). Autoimmune lesions had lower telomerase expression (mean+/-SE=1.25+/-0.5 relative intensity) compared to FTC (p=0.01), PTC (p=0.06) and benign lesions (p=0.15). Among PTC, 19 (70%) expressed telomerase, and 8 (30%) did not. Direct invasion (no.=4, 21%), distant metastasis (no.=2, 10%) and recurrence (no.=7, 37%) developed exclusively in PTC that expressed telomerase (p=0.02). Disease-free survival was also shorter for PTC that expressed telomerase (p=0.06). Recurrence developed in 1/2 (50%) FTC that expressed telomerase. We conclude that childhood thyroid cancers which express telomerase have an increased risk of tissue invasion, metastasis, and recurrence.

Serum vascular endothelial growth factor levels are elevated in metastatic differentiated thyroid cancer but not increased by short-term TSH stimulation.

Solid tumor formation requires the development of a blood supply adequate to meet the metabolic demands of the enlarging tumor mass that cannot be sustained by simple diffusion. One principal stimulant to endothelial cell growth and migration, vascular endothelial growth factor (VEGF), is synthesized and secreted by thyroid cancer cells. Furthermore, VEGF overexpression is associated with an aggressive thyroid cancer phenotype in both animal models and clinical-pathological studies. In other malignancies, elevated serum levels of VEGF often correlate with stage of disease and other poor prognostic clinical features. Therefore, we hypothesized that serum VEGF levels would be significantly higher in patients with persistent or recurrent thyroid cancer than in those cured of the disease. Because TSH stimulates both normal and neoplastic thyroid cells, we also proposed that serum VEGF would be further increased by TSH stimulation. Sixty-nine patients with either papillary or follicular thyroid cancer, status post total thyroidectomy, and prior radioactive iodine ablation, who had undergone routine recombinant human TSH (rhTSH, Thyrogen, Genzyme Transgenics Corp., Cambridge, MA) assisted whole-body radioactive iodine scanning, were included in this study. This cohort (mean age 53 +/- 16 yr, 51% female) included 21 patients with no evidence of disease and 48 patients with local or distant metastases. Stored serum samples obtained for standard Tg determinations before and 72 h following standard rhTSH stimulation were identified and assayed for VEGF 165 (R \[amp ]\ D Systems, Minneapolis, MN). Baseline serum VEGF levels obtained at a time of TSH suppression were significantly higher in patients with known metastatic disease than in those with no evidence of disease (416 +/- 62 pg/ml vs. 185 +/- 25 pg/ml, P = 0.001). Patients with distant metastases had baseline serum VEGF levels that did not differ significantly from patients with only cervical recurrences (455 +/- 90 pg/ml in distant metastases vs. 330 +/- 44 pg/ml for local cervical recurrences). Short-term TSH stimulation, although causing a significant rise in serum Tg, resulted in no significant increase in serum VEGF measured 72 h after rhTSH injection in either the patients with known metastatic disease (416 +/- 62 pg/ml baseline vs. 419 +/- 71 pg/ml after TSH stimulation) or in cured patients (185 +/- 25 pg/ml baseline vs. 191 +/- 33 pg/ml after TSH stimulation). Subgroup analysis revealed that patients with metastatic disease arising from well differentiated primary thyroid cancers had significantly higher serum VEGF levels than patients with metastatic disease arising from poorly differentiated thyroid cancer primaries (485 +/- 74 pg/ml vs. 167 +/- 32 pg/ml, P = 0.003 by ANOVA). Poorly differentiated metastatic thyroid cancers had serum VEGF levels indistinguishable from patients cured of disease (167 +/- 32 pg/ml vs. 186 +/- 25 pg/ml). In summary, serum VEGF is significantly elevated in patients with metastatic differentiated thyroid cancer but not in those with poorly differentiated thyroid cancer metastases. No measurable increase in serum VEGF levels can be detected 72 h after short-term TSH stimulation with rhTSH. We conclude that serum VEGF may serve as a clinical useful marker of residual differentiated thyroid cancer.

Nuclear localization of thyroid transcription factor-1 correlates with serum thyrotropin activity and may be increased in differentiated thyroid carcinomas with aggressive clinical course.

Thyroid transcription factor 1 (TTF-1) is essential for thyroid differentiation and regulates expression of thyroglobulin, thyroid peroxidase, sodium/iodide symporter, and thyrotropin receptor (TSH-R) genes. Because thyrotropin (TSH) upregulates these same genes, we hypothesized TSH-R activation might increase TTF-1 and that TTF-1 might be differentially expressed in benign and malignant thyroid disease. TTF-1 expression and sub-cellular localization were determined by immunohistochemistry in 62 thyroid carcinomas, 15 benign lesions, and 2 normal thyroids. Nuclear TTF-1 was detected in benign (77%) and malignant lesions (69%), with similar intensity in both (1.1+/-0.19 versus 1.0+/-0.10). Nuclear TTF-1 staining correlated with the effective serum TSH level (p = 0.02) and patient age (p < 0.05). Nuclear TTF-1 was detected in 35 papillary thyroid carcinomas (PTC), of which 23% developed recurrent or persistent disease, and was absent from 18 PTC, of which only 6% recurred (p = 0.06). We conclude that nuclear TTF-1 correlates with serum TSH activity, increases with age, and may be increased in persistent or recurrent PTC.

The effect of insulin-like growth factor analogs on turkey satellite cell and embryonic myoblast proliferation.

The effects of several human and chicken insulin-like growth factor (IGF) analogs on turkey satellite cell and embryonic myoblast proliferation were examined in serum-free medium. Similar rates of proliferation were observed when human or chicken IGF-I or IGF-II (13.1 nM) was administered to satellite cells. The biopotency of two analogs, which were modified to prevent interaction with IGF-binding proteins, was also examined. Human Des(1-6)IGF-II was equipotent to native human and chicken IGF-II. However, the chicken LR3 IGF-I analog was significantly less active toward satellite cells and embryonic myoblasts compared with chicken IGF-I. Human [Leu27] IGF-II, an analog designed to have reduced affinity to the IGF Type I receptor but unaltered binding to IGF-binding proteins, had a diminished effect on cell proliferation. Examination of IGF receptor binding characteristics revealed that chicken LR3 IGF-I had reduced ability to compete with [125I]hIGF-I for binding to satellite cells or embryonic myoblasts compared with chicken IGF-I. The observed biological responses to IGF suggest that IGF-binding proteins have little effect on Type I IGF receptor action in these cell types in serum-free medium. The results also suggest that alterations of the IGF molecule to prevent interaction with binding proteins may also alter receptor binding affinity.

Linkers for improved cleavage of fusion proteins with an engineered alpha-lytic protease.

Addition of an N-terminal fusion partner can greatly aid the expression and purification of a recombinant protein in Escherichia coli. We investigated two genetically engineered proteases designed to remove the fusion partner after the protein of interest has been expressed. Recombinant human insulin-like growth factor-II (hIGF-II) has been produced from E. coli-derived fusion proteins using a novel enzymatic cleavage system that uses a mutant of alpha-lytic protease. Initially, two potential fusion protein linkers were designed, Pro-Ala-Pro-His (PAPH) and Pro-Ala-Pro-Met (PAPM), and were tested as substrates in the form of synthetic dodecapeptides. Using mass spectrometry and reverse-phase HPLC, the position of cleavage was confirmed and the kinetics of synthetic peptide cleavage were examined. Use of the linkers in hIGF-II fusion proteins produced in E. coli was then evaluated. The fusion proteins constructed consist of the first 11 amino acids of porcine growth hormone linked N-terminally to hIGF-II by six amino acids that include the dipeptide Val-Asn followed by a variable tetrapeptide protease cleavage motif. Mass spectrometry and N-terminal sequencing confirmed that proteolytic cleavage of the fusion proteins had occurred at the predicted sites. Using the fusion proteins as substrates, the cleavage of the rationally designed motifs by the alpha-lytic protease mutant was compared. The fusion protein containing the motif PAPM had a k(cat)/K(M) ratio indicating a 1.6-fold preference over the PAPH fusion protein for cleavage by this enzyme. Furthermore, when hIGF-II fusion proteins containing the designed cleavable linkers were processed with the engineered alpha-lytic protease, they gave greatly improved yields of native hIGF-II compared to an analogous fusion protein cleaved by H64A subtilisin. Comparison of the peptide and protein cleavage studies shows that the efficient proteolysis of the cleavage motifs is an inherent property of the designed sequences and is not determined by secondary or tertiary structure in the fusion proteins.

Intensity of vascular endothelial growth factor expression is associated with increased risk of recurrence and decreased disease-free survival in papillary thyroid cancer.

BACKGROUND: Vascular endothelial growth factor (VEGF) induces proliferation of endothelial cells, stimulates angiogenesis, and increases vascular permeability. Increased VEGF expression has been associated with poor clinical outcomes in many malignancies. Several recent reports have documented over expression of VEGF in papillary thyroid cancer. We hypothesized that increased expression of VEGF would be associated with either an increased risk of recurrence or a decreased recurrence-free survival in papillary thyroid cancer. METHODS: Immunohistochemistry was used to detect VEGF expression in archival paraffin-embedded surgical thyroid specimens from 96 subjects with papillary thyroid cancer. RESULTS: VEGF expression was detected in 98% (94/96) of the samples, predominantly of slight-to-moderate intensity in the majority of malignant cells. However, the specific finding of a diffuse pattern of intense immunostaining for VEGF was detected significantly more often than less intense, patchy immunostaining patterns in subjects with distant metastasis at diagnosis (63% versus 15%, P =.005), local recurrence (58% versus 13%, P =.001), and distant recurrence (83% versus 14%, P =.001). Furthermore, this specific pattern of diffuse, intense VEGF expression was associated with a significantly shorter recurrence-free survival than other staining patterns (P =.007). CONCLUSIONS: These data demonstrate that the immunohistochemical pattern of VEGF staining in the initial surgical specimen is strongly associated with the incidence of local and distant metastasis in papillary thyroid cancer.

Thyroglobulin messenger ribonucleic acid levels in the peripheral blood of children with benign and malignant thyroid disease.

Reverse transcriptase-PCR has identified thyroglobulin mRNA (Tg mRNA) in peripheral blood of normal adults and adults with thyroid cancer. However, no children were studied. The primary objective of this study was to determine whether whole blood Tg mRNA levels differ between benign and malignant thyroid disease in children. The secondary goals were to determine whether whole blood Tg mRNA levels vary with age or pubertal development among children with thyroid disease. Whole blood Tg mRNA levels were determined in 38 children (29 girls, nine boys; median age, 14.5 y; range, 4.8-20.4 y) with benign and malignant thyroid disease and correlated with diagnosis, age, pubertal status, thyroid size, and serum levels of free thyroxine, TSH, and Tg protein. Tg mRNA levels ranged from 3.3 to 104 pg Eq/microg total thyroid RNA (mean, 28 +/- 20.2 pg Eq/microg total thyroid RNA) and were similar in benign and malignant disorders (p = 0.67). However, in children with previously treated papillary thyroid cancer, Tg mRNA levels directly correlated with total body (131)I uptake (p = 0.026) and serum Tg protein (p = 0.037). There was no difference between boys and girls, and no change with pubertal maturation. In children with benign thyroid disease, Tg mRNA levels correlated with serum TSH (p = 0.031), but not with diagnosis, age, Tanner stage, or thyroid size. We conclude that Tg mRNA levels are similar in children with benign and malignant thyroid disease and unchanged by age or pubertal status, but correlated with tumor burden in previously treated papillary thyroid cancer.

Infiltration of differentiated thyroid carcinoma by proliferating lymphocytes is associated with improved disease-free survival for children and young adults.

An immune response directed against thyroid cancer might be important in preventing metastasis and recurrence. This idea is supported by previous observations showing that adults with autoimmune thyroiditis or lymphocytic infiltration surrounding papillary thyroid carcinoma (PTC) have improved disease-free survival. The long-term outcome for differentiated thyroid cancer is even more favorable for children and young adults. If the immune response is important, we hypothesized that tumor-associated lymphocytes with a high proliferation index would be found in thyroid cancers from children and young adults and would be associated with improved disease-free survival. Using immunohistochemistry, we examined 39 childhood PTC, 9 follicular thyroid carcinomas, 2 medullary thyroid carcinomas, 11 benign thyroid lesions, and 2 normal thyroid glands for the presence of lymphocytes (leukocyte common antigen) and lymphocyte proliferation (proliferating cell nuclear antigen, Ki-67). The majority of PTC (65%) and follicular thyroid carcinomas (75%) from children and young adults contained lymphocytes in the immediate vicinity of thyroid cancers, but only 7 (18%) patients with PTC also had a diagnosis of autoimmune thyroiditis. Disease-free survival did not correlate with the presence or number of lymphocytes per high power field. In contrast, disease-free survival was significantly improved (P = 0.01) for thyroid cancers with the greatest number of Ki-67-positive lymphocytes per high power field. The number of lymphocytes per high powered field was greater for multifocal PTC (P: = 0.023), and the number of proliferating lymphocytes was greatest for PTC with regional lymph node involvement (30.5 +/- 12.3 vs. 6.8 +/- 5.0; P = 0.047). We conclude that proliferation of tumor-associated lymphocytes is associated with improved disease-free survival for children and young adults with thyroid cancer.

Over-expression of hepatocyte growth factor/scatter factor (HGF/SF) and the HGF/SF receptor (cMET) are associated with a high risk of metastasis and recurrence for children and young adults with papillary thyroid carcinoma.

OBJECTIVE: The study determined if hepatocyte growth factor/scatter factor (HGF/SF) or the HGF/SF receptor (cMET) might be important for metastasis in thyroid cancer. DESIGN: We examined HGF/SF and cMET expression by immunohistochemistry in a retrospective group of benign and malignant thyroid lesions from children and young adults, and correlated the intensity of expression with clinical outcome. PATIENTS: Patients included 42 children and young adults with papillary thyroid carcinomas (PTC), seven with follicular thyroid carcinomas (FTC), two with medullary thyroid carcinomas (MTC), 14 with benign thyroid disorders, and two with normal thyroids. MEASUREMENTS: Expression of cMET was graded from 0 (absent) to 4 (intense); and HGF/SF expression was graded from 0 (absent-minimal) to 3 (diffuse and intense). RESULTS: cMET staining was greater in PTC (mean intensity 2.3 +/- 0.4 vs. 0.8 +/- 0.2, P < 0.005) and FTC (2.4 +/- 0.6 vs. 0.8 +/- 0.2, P = 0.04) than benign lesions (0.8 +/- 0.2) or normal thyroids (0.4 +/- 0.5). PTC with intense cMET staining had shorter disease free survival (P = 0.05) and increased HGF/SF staining (r = 0.39, P = 0.017). HGF/SF correlated with the extent of disease at diagnosis (r = 0.33, P = 0.049). Patients with PTC were stratified into quartiles based on combined cMET and HGF/SF staining. Those with intense cMET and HGF/SF staining were younger (P = 0.05), and had reduced disease free survival (P = 0.03). CONCLUSIONS: We conclude that increased cMET and HGF/SF expression is associated with a high risk for metastasis and recurrence in children and young adults with papillary thyroid carcinoma.

The expression of vascular endothelial growth factor and the type 1 vascular endothelial growth factor receptor correlate with the size of papillary thyroid carcinoma in children and young adults.

Vascular endothelial growth factor (VEGF) is essential for the growth of many solid tumors, but there are little data regarding VEGH in childhood thyroid cancers. We examined the relationships between VEGF, the type 1 VEGF receptor (FLT-1) and clinical outcome for a group of thyroid cancers in children and young adults. The expression of VEGF and FLT-1 were determined by immunohistochemistry using archival, paraffin-embedded thyroid tissue blocks and compared with the retrospective clinical outcome for each patient. The study included 67 children and young adults with papillary thyroid carcinoma (PTC, n = 42), follicular thyroid carcinoma (FTC, n = 8), benign lesions (n = 15), or controls (n = 2). VEGF expression was greater in PTC (mean intensity 2.23 +/- 0.25, p = 0.002) and FTC (2.8 +/- 0.73, p = 0.01) than benign lesions (1.0 +/- 0.27), and correlated with PTC size (r = 0.42, p = 0.008). FLT-1 expression was greater in PTC (mean intensity 2.8 +/- 0.17) than FTC (1.9 +/- 0.25, p = 0.015) and benign lesions (1.7 +/- 0.32, p = 0.002); and correlated with PTC size (r = 0.41, p = 0.01) as well as VEGF expression (r = 0.52, p = 0.002). Recurrent disease developed exclusively in patients with PTC which expressed VEGF (7/28, 95% CI 10.6%-44.2%). PTC that did not express VEGF (0/8, 95% CI = 0%-31.2%) did not recur; however, the difference was not statistically significant (p = 0.15). We conclude that the expression of VEGF and FLT-1 are directly correlated with the size of PTC in children and young adults.

Autoantibodies to p53 in sera of patients with autoimmune thyroid disease.

Mutations in the tumor suppressor gene, p53, lead to intracellular accumulation of abnormal p53 protein and are associated with p53 autoantibodies. p53 also accumulates in autoimmune diseases and Hashimoto's thyroiditis, but it is unknown if p53 autoantibodies occur in the latter. We measured p53 autoantibodies in the sera of 93 patients with thyroid disease and 19 patients without thyroid disease. Anti-p53 antibodies were detected in the sera from 4.2% (2/48) of patients with autoimmune thyroid disease, including one patient with Hashimoto's thyroiditis (3.7%, 1/27) and one with Graves' disease (4.8%, 1/21). A third patient with pseudohypoparathyroidism, but without thyroid disease, was also positive (1/19; 5.2%). None of 19 patients with differentiated thyroid cancer had anti-p53 antibodies. We conclude that anti-p53 antibodies can be detected in the sera from approximately 4% of patients with autoimmune thyroid disease. This finding suggests that increased DNA damage and apoptosis may be associated with autoimmune thyroid disease.

Solution structure and backbone dynamics of long-[Arg(3)]insulin-like growth factor-I.

Long-[Arg(3)]insulin-like growth factor-I (IGF-I) is a potent analog of insulin-like growth factor-I that has been modified by a Glu(3) --> Arg mutation and a 13-amino acid extension appended to the N terminus. We have determined the solution structure of (15)N-labeled Long-[Arg(3)]-IGF-I using high resolution NMR and restrained molecular dynamics techniques to a precision of 0.82 +/- 0.28 A root mean square deviation for the backbone heavy atoms in the three alpha-helices and 3.5 +/- 0.9 A root mean square deviation for all backbone heavy atoms excluding the 8 N-terminal residues and the 8 C-terminal eight residues. Overall, the structure of the IGF-I domain is consistent with earlier studies of IGF-I with some minor changes remote from the N terminus. The major variations in the structure, compared with IGF-I, occur at the N terminus with a substantial reorientation of the N-terminal three residues of the IGF-I domain. These results are interpreted in terms of the lower binding affinity for insulin-like growth factor-binding proteins. The backbone dynamics of Long-[Arg(3)]IGF-I were investigated using (15)N nuclear spin relaxation and the heteronuclear nuclear Overhauser enhancement (NOE). There is a considerable degree of flexibility in Long-[Arg(3)]IGF-I, even in the alpha-helices, as indicated by an average ((1)H)(15)N NOE of 0.55 for the regions. The largest heteronuclear NOEs are observed in the helical regions, lower heteronuclear NOEs are observed in the C-domain loop separating helix 1 from helix 2, and negative heteronuclear NOEs are observed in the N-terminal extension and at the C terminus. Despite these data indicating conformational flexibility for the N-terminal extension, slow amide proton exchange was observed for some residues in this region, suggesting some transitory structure does exist, possibly a molten helix. A certain degree of flexibility may be necessary in all insulin-like growth factors to enable association with various receptors and binding proteins.

The ret/PTC mutations are common in sporadic papillary thyroid carcinoma of children and young adults.

The ret/PTC rearrangements (PTC-1, PTC-2, and PTC-3) are characteristic of papillary thyroid cancer (PTC). In adults, PTC-1 is common and may be associated with an aggressive clinical course. The incidence and significance of ret/PTC mutations are less well understood in children. We examined spontaneous PTC from 33 patients (23 females and 10 males) with a median age of 18 yr (range, 6-21 yr) and a median follow-up of 3.5 yr (range, 0-13.4 yr). The ret/PTC mutations were identified in 15 tumors (45%), including 8 PTC-1 (8 of 15, 53%), 2 PTC-2 (2 of 15, 13%), 2 PTC-3 (2 of 15, 13%), and 3 (3 of 15, 20%) combined PTC mutations (PTC-1 and PTC-2). This distribution is significantly different (P = 0.001, by chi2 analysis) from that reported for children with radiation-induced PTC. There was no correlation between the presence or type of ret/PTC mutation and patient age, tumor size, focality, extent of disease at diagnosis, or recurrence. We conclude that ret/PTC mutations are 1) common in sporadic childhood PTC, 2) predominantly PTC-1, 3) frequently multiple, and 4) of different distribution than that reported for children with radiation-induced PTC.

Ras mutations are uncommon in sporadic thyroid cancer in children and young adults.

Mutations in the ras genes (H-ras, K-ras, and N-ras) occur in 10-15% of all human cancers, and commonly arise from single base substitutions at codons 12, 13, or 61. Although ras mutations have been found in adult thyroid cancers, they were absent from the two studies which examined childhood thyroid cancers. Both studies included only children with radiation induced thyroid cancer, and it remains unclear if ras mutations occur in children without radiation exposure. To answer this question, we examined archival tissue blocks from 31 children with papillary thyroid cancer (PTC) 4 with follicular thyroid cancer (FTC), 2 with medullary thyroid cancer (MTC), and 1 with lymphoma (LYM). Only 1 patient with PTC had previous radiation exposure. Genomic DNA was extracted and used for PCR amplification of the ras genes. The PCR products were analyzed by oligospecific hybridization for mutations at codons 12, 13, and 61. Two of the PTCs (6.5%) contained ras mutations. Both patients had class II disease and no history of previous radiation exposure. One patient subsequently developed bone and lung metastases. The patient with lymphoma also had a ras mutation (N-61), but ras mutations were absent from all FTC and MTC. These results suggest that ras mutations are uncommon in spontaneous childhood thyroid cancer, but occur with a frequency similar to that found in previous reports of adult differentiated thyroid cancers. The number of subjects was too small to determine if ras mutations are more common in patients with aggressive papillary thyroid cancer.

Quantitative reverse transcription-polymerase chain reaction of circulating thyroglobulin messenger ribonucleic acid for monitoring patients with thyroid carcinoma.

Patients with thyroid cancer are monitored for disease recurrence by measurement of serum thyroglobulin (Tg) and iodine-131 (131I) scanning. To enhance sensitivity and to circumvent antibodies that interfere with Tg immunoassays, we have developed RT-PCR assays that detect circulating thyroid messenger RNA (mRNA) transcripts. We now report results using a sensitive quantitative Tg mRNA assay (Taqman; ABI, Foster City, CA) in comparison with immunoassay in patients previously treated for thyroid cancer. We evaluated 107 patients: 84 during T4 therapy, 14 after T4 withdrawal, and 9 at both time points. All patients had near-total thyroidectomy, and 92% received postoperative 131I. Serum TSH, Tg protein, and Tg mRNA were measured. Patients were grouped based on most recent 131I scan or pathologically confirmed disease as having no detectable thyroid tissue (n = 33), thyroid bed uptake (n = 37), cervical/regional adenopathy (n = 21), or distant metastases (n = 16). During T4 therapy, median (range) Tg mRNA values (pg Tg Eq/microg thyroid RNA) for the groups were 1.5 (0-26.8), 9.4 (0.5-90.0), 15.4 (0.2-92), and 12.4 (1.9-16.6), respectively. Using a value of 3 pg Tg Eq/microg thyroid RNA as cut-point, Tg mRNA was positive in 38% of patients with no uptake, 75% with thyroid bed uptake, 84% with cervical/regional disease, and 94% with distant metastases. The median Tg mRNA value for patients with no uptake was lower than the median values for patients with thyroid bed uptake (P = 0.009) or with detectable thyroid tissue at any site (P = 0.010). Patients with negative 131I whole body scans were also less likely to have detectable Tg mRNA levels than were patients with thyroid bed uptake (P < 0.001) or any detectable thyroid tissue at any location (P < 0.001). Similar differences between these groups were seen after T4 withdrawal and for the 23 patients with circulating anti-Tg antibodies, when analyzed separately. Eight of the nine patients studied with low and high TSH concentrations displayed greater amounts of circulating Tg mRNA after T4 withdrawal. In three patients followed prospectively, the amount Tg mRNA correlated with the presence and absence of cervical metastases. In conclusion, we have demonstrated that a quantitative Tg mRNA assay can identify thyroid cancer patients with recurrent or residual thyroid tissue with greater sensitivity and similar specificity to Tg immunoassay during T4 therapy. The assay was unaffected by anti-Tg antibodies, responded to TSH-stimulation, and was reduced after surgical removal of metastases. These data suggest that this quantitative Tg mRNA assay may be a sensitive marker of tumor recurrence or response to therapy, particularly in patients with anti-Tg antibodies.

Update on the genetics and pathophysiology of type I diabetes mellitus.

Type I diabetes mellitus results from the genetically predetermined autoimmune destruction of pancreatic beta-cells, resulting in gradual, but complete, loss of insulin secretion. There are strong associations with specific HLA haplotypes, but environmental triggers are also required to initiate beta-cell autoimmunity. These could possibly include enteroviral infection, early weaning from breastfeeding, early exposure to cow's milk antigens, and free radical damage. Once initiated, beta-cell autoimmunity does not always lead to clinical diabetes, suggesting that immunomodulators may be important in the control of beta-cell destruction. Current interventions designed to prevent type I diabetes mellitus are based on attempts to alter this immune response and to preserve beta-cell function. It is important for the pediatrician to understand the background of these trials and to be able to answer parents' questions regarding study participation.

How do the results of the diabetes control and complications trial relate to the practice of pediatrics: who should have intensive management?

The DCCT showed that any improvement in glycemic control decreases the risk of long-term complications. Although expensive, time consuming, and associated with increased risks of hypoglycemia and obesity, improved glycemic control is of benefit as long as hypoglycemia is avoided. Specific HbA1c target levels must be individualized and age appropriate.